The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study is protein kinases.
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (See, Hardie, G. and Hanks, S. The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576-596; Knighton et al., Science 1991, 253, 407-414; Hiles et al., Cell 1992, 70, 419-429; Kunz et al., Cell 1993, 73, 585-596; Garcia-Bustos et al., EMBO J. 1994, 13, 2352-2361).
In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease, and hormone-related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.
Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases consisting of a β-sheet rich amino-terminal lobe and a larger carboxy-terminal lobe that is largely α-helical. The CDKs display the 11 subdomains shared by all protein kinases and range in molecular mass from 33 to 44 kD. This family of kinases, which includes CDK1, CKD2, CDK4, and CDK6, requires phosphorylation at the residue corresponding to CDK2 Thr160 in order to be fully active [Meijer, L., Drug Resistance Updates 2000, 3, 83-88].
Each CDK complex is formed from a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytic kinase subunit (e.g., CDK1, CDK2, CDK4, CDK5, and CDK6). Each different kinase/cyclin pair functions to regulate the different and specific phases of the cell cycle known as the G1, S, G2, and M phases [Nigg, E., Nature Reviews 2001, 2, 21-32; Flatt, P., Pietenpol, J., Drug Metabolism Reviews 2000, 32, 283-305].
The CDKs have been implicated in cell proliferation disorders, particularly in cancer. Cell proliferation is a result of the direct or indirect deregulation of the cell division cycle and the CDKs play a critical role in the regulation of the various phases of this cycle. For example, the over-expression of cyclin D1 is commonly associated with numerous human cancers including breast, colon, hepatocellular carcinomas and gliomas [Flatt, P., Pietenpol, J., Drug Metabolism Reviews 2000, 32, 283-305]. The CDK2/cyclin E complex plays a key role in the progression from the early G1 to S phases of the cell cycle and the overexpression of cyclin E has been associated with various solid tumors. Therefore, inhibitors of cyclins D1, E, or their associated CDKs are useful targets for cancer therapy [Kaubisch, A., Schwartz, G., The Cancer Journal 2000, 6, 192-212].
CDKs, especially CDK2, also play a role in apoptosis and T-cell development. CDK2 has been identified as a key regulator of thymocyte apoptosis [Williams, O. et al., European Journal of Immunology 2000, 709-713]. Stimulation of CDK2 kinase activity is associated with the progression of apoptosis in thymocytes, in response to specific stimuli. Inhibition of CDK2 kinase activity blocks this apoptosis resulting in the protection of thymocytes.
In addition to regulating the cell cycle and apoptosis, the CDKs are directly involved in the process of transcription. Numerous viruses require CDKs for their replication process. Examples where CDK inhibitors restrain viral replication include human cytomegalovirus, herpes virus, and varicella-zoster virus [Meijer, L., Drug Resistance Updates 2000, 3, 83-88].
Inhibition of CDK is also useful for the treatment of neurodegenerative disorders such as Alzheimer's disease. The appearance of Paired Helical Filaments (PHF), associated with Alzheimer's disease, is caused by the hyperphosphorylation of Tau protein by CDK5/p25 [Meijer, L., Drug Resistance Updates, 2000 3, 83-88].
The ribosomal protein kinases p70S6K-1 and -2 are members of the AGC sub-family of protein kinases that consists of, amongst others, PKB and MSK. The p70S6 kinases catalyze the phosphorylation and subsequent activation of the ribosomal protein S6, which has been implicated in the translational up-regulation of mRNAs coding for the components of the protein synthetic apparatus.
These mRNAs contain an oligopyrimidine tract at their 5′ transcriptional start site, termed a 5′TOP, which has been shown to be essential for their regulation at the translational level (Volarevic, S. et al., Prog. Nucleic Acid Res. Mol. Biol. 2001, 65, 101-186). p70 S6K dependent S6 phosphorylation is stimulated in response to a variety of hormones and growth factors primarily via the P13K pathway (Coffer, P. J. et al., Biochem. Biophys. Res. Commun., 1994 198, 780-786), which maybe under the regulation of mTOR, since rapamycin acts to inhibit p70S6K activity and blocks protein synthesis, specifically as a result of a down-regulation of translation of these mRNA's encoding ribosomal proteins (Kuo, C. J. et al., Nature 1992, 358, 70-73).
In vitro PDK1 catalyses the phosphorylation of Thr252 in the activation loop of the p70 catalytic domain, which is indispensable for p70 activity (Alessi, D. R., Curr. Biol., 1998, 8, 69-81). The use of rapamycin and gene deletion studies of dp70S6K from Drosophila and p70S6K1 from mouse have established the central role p70 plays in both cell growth and proliferation signaling.
A family of type III receptor tyrosine kinases including Flt3, c-Kit, PDGF-receptor and c-Fms play an important role in the maintenance, growth and development of hematopoietic and non-hematopoietic cells. [Scheijen, B., Griffin J. D., Oncogene, 2002, 21, 3314-3333 and Reilly, J. T., British Journal of Haematology, 2002, 116, 744-757]. FLT-3 and c-Kit regulate maintenance of stem cell/early progenitor pools as well the development of mature lymphoid and myeloid cells [Lyman, S., Jacobsen, S., Blood, 1998, 91, 1101-1134]. Both receptors contain an intrinsic kinase domain that is activated upon ligand-mediated dimerization of the receptors. Upon activation, the kinase domain induces autophosphorylation of the receptor as well as the phosphorylation of various cytoplasmic proteins that help propagate the activation signal leading to growth, differentiation and survival. Some of the downstream regulators of FLT-3 and c-Kit receptor signaling include, PLCγ, PI3-kinase, Grb-2, SHIP and Src related kinases [Scheijen, B., Griffin J. D., Oncogene, 2002, 21, 3314-3333]. Both receptor tyrosine kinases have been shown to play a role in a variety of hematopoietic and non-hematopoietic malignancies. Mutations that induce ligand independent activation of FLT-3 and c-Kit have been implicated acute-myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), mastocytosis and gastrointestinal stromal tumor (GIST). These mutations include single amino acid changes in the kinase domain or internal tandem duplications, point mutations or in-frame deletions of the juxtamembrane region of the receptors. In addition to activating mutations, ligand dependent (autocrine or paracrine) stimulation of over-expressed wild-type FLT-3 or c-Kit can contribute to the malignant phenotype [Scheijen, B., Griffin J. D., Oncogene, 2002, 21, 3314-3333].
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase comprised of α and β isoforms that are each encoded by distinct genes [Coghlan et al., Chemistry & Biology, 2000 7, 793-803; Kim and Kimmel, Curr. Opinion Genetics Dev., 2000, 10, 508-514]. GSK-3 has been implicated in various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative diseases, and cardiomyocyte hypertrophy [see, e.g., WO 99/65897; WO 00/38675; Kaytor and Orr, Curr. Opin. Neurobiol., 2000, 12, 275-8; Haq et al., J. Cell Biol., 2000, 151, 117-30; Eldar-Finkelman, Trends Mol. Med., 2000, 8, 126-32]. These diseases are associated with the abnormal operation of certain cell signaling pathways in which GSK-3 plays a role.
GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. These include glycogen synthase, which is the rate-limiting enzyme required for glycogen synthesis, the microtubule-associated protein Tau, the gene transcription factor β-catenin, the translation initiation factor e1F-2B, as well as ATP citrate lyase, axin, heat shock factor-1, c-Jun, c-myc, c-myb, CREB, and CEPBα. These diverse targets implicate GSK-3 in many aspects of cellular metabolism, proliferation, differentiation and development.
In a GSK-3 mediated pathway that is relevant for the treatment of type II diabetes, insulin-induced signaling leads to cellular glucose uptake and glycogen synthesis. GSK-3 is a negative regulator of the insulin-induced signal in this pathway. Normally, the presence of insulin causes inhibition of GSK-3-mediated phosphorylation and deactivation of glycogen synthase. The inhibition of GSK-3 leads to increased glycogen synthesis and glucose uptake [Klein et al., PNAS, 1996, 93, 8455-9; Cross et al., Biochem. J., 1994, 303, 21-26; Cohen, Biochem. Soc. Trans., 1993, 21, 555-567; and Massillon et al., Biochem. J. 1994, 299, 123-128; Cohen and Frame, Nat. Rev. Mol. Cell. Biol., 2001, 2, 769-76]. However, where the insulin response is impaired in a diabetic patient, glycogen synthesis and glucose uptake fail to increase despite the presence of relatively high blood levels of insulin. This leads to abnormally high blood levels of glucose with acute and chronic effects that may ultimately result in cardiovascular disease, renal failure and blindness. In such patients, the normal insulin-induced inhibition of GSK-3 fails to occur. It has also been reported that GSK-3 is overexpressed in patients with type II diabetes [WO 00/38675]. Therapeutic inhibitors of GSK-3 are therefore useful for treating diabetic patients suffering from an impaired response to insulin.
Apoptosis has been implicated in the pathophysiology of ischemic brain damage (Li et al., 1997; Choi, et al., 1996; Charriaut-Marlangue et al., 1998; Grahm and Chen, 2001; Murphy et al., 1999; Nicotera et al., 1999). Recent publications indicate that activation of GSK-3β may be involved in apoptotic mechanisms (Kaytor and Orr, 2002; Culbert et al., 2001). Studies in rat models of ischemic stroke induced by middle cerebral artery occlusion (MCAO) showed increased GSK-3β expression is following ischemia (Wang et al., Brain Res., 2000, 859, 381-5; Sasaki et al., Neurol Res., 2001, 23, 588-92). Fibroblast growth factor (FGF) reduced ischemic brain injury after permanent middle cerebral artery occlusion (MCO) in rats (Fisher et al. 1995; Song et al. 2002). Indeed, the neuroprotective effects of FGF demonstrated in ischemia models in rats may be mediated by a PI-3 kinase/AKT-dependent inactivation of GSK-3β (Hashimoto et al., 2002). Thus, inhibition of GSK-3β after a cerebral ischemic event may ameliorate ischemic brain damage.
GSK-3 is also implicated in mycardial infarction. See Jonassen et al., Circ. Res., 2001, 89, 1191 (the reduction in myocardial infarction by insulin administration at reperfusion is mediated via Akt dependent signaling pathway.); Matsui et al., Circulation, 2001, 104, 330 (Akt activation preserves cardiac finction and prevents cardiomyocyte injury after transient cardiac ischemia in vivo); Miao et al., J. Mol. Cell. Cardiol., 2000, 32, 2397 (intracoronary, adenovirus-mediated Akt gene delivery in heart reduced gross infarct size following ischemia-reperfusion injury in vivo); and Fujio et al., Circulation, 2000, 101, 660 (Akt signaling inhibits cardiac myocyte apoptosis in vitro and protects against ischemia-reperfusion injury in mouse heart).
GSK-3 activity plays a role in head trauma. See Noshita et al., Neurobiol. Dis., 2002, 9, 294 (upregulation of Akt/PI3-kinase pathway may be crucial for cell survival after traumatic brain injury) and Dietrich et al., J. Neurotrauma, 1996, 13, 309 (posttraumatic administration of bFGF significantly reduced damaged cortical neurons & total contusion volume in a rat model of traumatic brain injury).
GSK-3 is also known to play a role in psychiatric disorders. See Eldar-Finkelman, Trends Mol. Med., 2002, 8, 126; Li et al., Bipolar Disord., 2002, 4, 137 (LiCl and Valproic acid, anti-psychotic, mood stabilizing drugs, decrease GSK-3 activities and increase beta-catenin) and Lijam et al., Cell, 1997, 90, 895 (dishevelled KO mice showed abnormal social behavior and defective sensorimotor gating. Dishevelled, a cytoplamic protein involved in WNT pathway, inhibits GSK-3beta activities).
It has been shown that GSK-3 inhibition by lithium and valproic acid induces axonal remodeling and change synaptic connectivity. See Kaytor & Orr, Curr. Opin. Neurobiol., 2002, 12, 275 (downregulation of GSK-3 causes changes in microtubule-associated proteins: tau, MAP1 & 2) and Hall et al., Mol. Cell. Neurosci., 2002, 20, 257 (lithium and valproic acid induces the formation of growth cone-like structures along the axons).
GSK-3 activity is also associated with Alzheimer's disease. This disease is characterized by the presence of the well-known β-amyloid peptide and the formation of intracellular neurofibrillary tangles. The neurofibrillary tangles contain hyperphosphorylated Tau protein, in which Tau is phosphorylated on abnormal sites. GSK-3 has been shown to phosphorylate these abnormal sites in cell and animal models. Furthermore, inhibition of GSK-3 has been shown to prevent hyperphosphorylation of Tau in cells [Lovestone et al., Curr. Biol., 1994, 4, 1077-86; and Brownlees et al., Neuroreport, 1997, 8, 3251-55; Kaytor and Orr, Curr. Opin. Neurobiol., 2000, 12, 275-8]. In transgenic mice overexpressing GSK-3, a significant increase in Tau hyperphosphorylation and abnormal morphology of neurons was observed [Lucas et al., EMBO J., 2001, 20, 27-39]. Active GSK-3 accumulates in cytoplasm of pretangled neurons, which can lead to neurofibrillary tangles in brains of patients with AD [Pei et al., J. Neuropathol. Exp. Neurol., 1999, 58, 1010-19]. Therefore, inhibition of GSK-3 slows or halts the generation of neurofibrillary tangles and thus can treat or reduce the severity of Alzheimer's disease.
Evidence for the role GSK-3 plays in Alzheimer's disease has been shown in vitro. See Aplin et al., J. Neurochem. 1996, 67, 699; Sun et al., Neurosci. Lett. 2002, 321, 61 (GSK-3b phosphorylates cytoplasmic domain of Amyloid Precursor Protein (APP) and GSK-3b inhibition reduces Ab40 & Ab42 secretion in APP-transfected cells); Takashima et al., PNAS, 1998, 95, 9637 (1998); Kirschenbaum et al., J. Biol. Chem., 2001, 276, 7366 (GSK-3b complexes with and phosphorylates presenilin-1, which is associated with gamma-secretase activity in the synthesis of Aβ from APP); Takashima et al., Neurosci. Res. 1998, 31, 317 (activation of GSK-3b by Ab(25-35) enhances phosphorylation of tau in hippocampal neurons. This observation provides a link between Aβ and neurofibrillary tangles composed of hyperphosphorylated tau, another pathological hallmark of AD); Takashima et al., PNAS, 1993, 90, 7789 (blockade of GSK-3b expression or activity prevents Ab-induced neurodegeneration of cortical and hippocampal primary cultures); Suhara et al., Neurobiol. Aging, 2003, 24, 437 (intracellular Ab42 is toxic to endothelial cells by interfering with activation of the Akt/GSK-3b signaling-dependent mechanism); De Ferrari et al., Mol. Psychiatry, 2003, 8, 195 (lithium protects N2A cells & primary hippocampal neurons from Aβ fibril-induced cytotoxicity, & reduces nuclear translocation/destabilization of b-catenin); and Pigino et al., J. Neurosci., 2003, 23, 4499 (the mutations in Alzheimer's presenilin 1 may deregulate and increase GSK-3 activity, which in turn, impairs axonal transport in neurons. The consequent reductions in axonal transport in affected neurons can ultimately lead to neurodegeneration).
Evidence for the role GSK-3 plays in Alzheimer's disease has been shown in vivo. See Yamaguchi et al., Acta Neuropathol., 1996, 92, 232; Pei et al., J. Neuropath. Exp. Neurol. 1999, 58, 1010 (GSK-3b immunoreactivity is elevated in susceptible regions of AD brains); Hernandez et al., J. Neurochem., 2002, 83, 1529 (transgenic mice with conditional GSK-3b overexpression exhibit cognitive deficits similar to those in transgenic APP mouse models of AD); De Ferrari et al., Mol. Psychiatry, 2003, 8, 195 (chronic lithium treatment rescued neurodegeneration and behavioral impairments (Morris water maze) caused by intrahippocampal injection of Aβ fibrils.); McLaurin et al., Nature Med., 2002, 8, 1263 (Immunization with Aβ in a transgenic model of AD reduces both AD-like neuropathology and the spatial memory impairments); and Phiel et al., Nature, 2003, 423, 435 (GSK-3 regulates amyloid-beta peptide production via direct inhibition of gamma secretase in AD tg mice).
Presenilin-1 and kinesin-1 are also substrates for GSK-3 and relate to another mechanism for the role GSK-3 plays in Alzheimer's disease, as was recently described by Pigino, G., et al., Journal of Neuroscience, 2003, 23, 4499. It was found that GSK-3beta phosphorylates kinsesin-I light chain, which results in a release of kinesin-1 from membrane-bound organelles, leading to a reduction in fast anterograde axonal transport (Morfini et al., 2002). The authors suggest that the mutations in PS1 may deregulate and increase GSK-3 activity, which in turn, impairs axonal transport in neurons. The consequent reductions in axonal transport in affected neurons ultimately leads to neurodegeneration.
GSK-3 is also associated with amyotrophic lateral sclerosis (ALS). See Williamson and Cleveland, 1999 (Axonal transport is retarded in a very early phase of ALS in mSOD1 mice); Morfini et al., 2002 (GSK3 phosphorylates kinesin light chains and inhibit anterograde axonal transport); Warita et al., Apoptosis, 2001, 6, 345 (2001) (The majority of spinal motor neurons lost the immunoreactivities for both PI3-K and Akt in the early and presymptomatic stage that preceded significant loss of the neurons in this SOD1 tg animal model of ALS); and Sanchez et al., 2001 (The inhibition of PI-3K induces neurite retraction mediated by GSK-3 activation).
GSK-3 activity is also linked to spinal cord and peripheral nerve injuries. It has been shown that GSK-3 inhibition by lithium and valproic acid can induce axonal remodeling and change synaptic connectivity. See Kaytor & Orr, Curr. Opin. Neurobiol., 2002, 12, 275 (downregulation of GSK-3 causes changes in microtubule-associated proteins: tau, MAP1 & 2) and Hall et al., Mol. Cell. Neurosci., 2002, 20, 257 (lithium and valproic acid induces the formation of growth cone-like structures along the axons). See also Grothe et al., Brain Res., 2000, 885, 172 (FGF-2 stimulates Schwann cell proliferation and inhibits myelination during axonal growth); Grothe and Nikkhah, 2001 (FGF-2 is up regulated in the proximal and distal nerve stumps within 5 hours after nerve crush); and Sanchez et al., 2001 (The inhibition of PI-3K induces neurite retraction mediated by GSK-3 activation).
Another substrate of GSK-3 is β-catenin, which is degraded after phosphorylation by GSK-3. Reduced levels of β-catenin have been reported in schizophrenic patients and have also been associated with other diseases related to increase in neuronal cell death [Zhong et al., Nature, 1998, 395, 698-702; Takashima et al., PNAS, 1993, 90, 7789-93; Pei et al., J. Neuropathol. Exp., 1997, 56, 70-78; and Smith et al., Bioorg. Med. Chem. 2001, 11, 635-639]. Furthermore, β-catenin and Tcf-4 play a dual role in vascular remodeling by inhibiting vascular smooth muscle cell apoptosis and promoting proliferation (Wang et al., Circ. Res., 2002, 90, 340. Accordingly, GSK-3 is associated with angiogenic disorders. See also Liu et al., FASEB J., 2002, 16, 950 (activation of GSK-3 reduces hepatocyte growth factor, leading to altered endothelial cell barrier function and diminished vascular integrity.) and Kim et al., J. Biol. Chem., 2002, 277, 41888 (GSK-3beta activation inhibits angiogenesis in vivo using a Matrigel plug assay: the inhibition of GSK-3beta signalling enhances capillary formation).
Association between GSK-3 and Huntington's disease has been shown. See Carmichael et al., J. Biol. Chem., 2002, 277, 33791 (GSK-3beta inhibition protect cells from poly-glutamine-induced neuronal and non-neuronal cell death via increases in b-catenin and its associated transcriptional pathway). Overexpression of GSK-3 reduced the activation of heat shock transcription factor-1 and heat shock protein HSP70 (Bijur et al., J. Biol. Chem., 2000, 275, 7583 that are shown to decrease both poly-(Q) aggregates and cell death in in vitro HD model (Wyttenbach et al., Hum. Mol. Genet., 2002, 11, 1137).
GSK-3 effects the levels of FGF-2 and their receptors which are increased during remyelination of brain aggregate cultures in remyelinating rat brains. See Copelman et al., 2000, Messersmith, et al., 2000; and Hinks and Franklin, 2000. It was also found that FGF-2 induces process outgrowth by oligodendrocytes implicating involvement of FGF in remyelination (Oh and Yong, 1996; Gogate et al., 1994) and that FGF-2 gene therapy has shown to improve the recovery of experimental allergic encephalomyelitis (EAE) mice (Ruffini, et al., 2001).
GSK-3 has also been associated with hair growth because Wnt/beta-catenin signaling is shown to play a major role in hair follicle morphogenesis and differentiation (Kishimotot et al., Genes Dev., 2000, 14, 1181; Millar, J. Invest. Dermatol., 2002, 118, 216). It was found that mice with constituitive overexpression of the inhibitors of Wnt signaling in skin failed to develop hair follicles. Wnt signals are required for the initial development of hair follicles and GSK-3 constituitively regulates Wnt pathways by inhibiting beta-catenin. (Andl et al., Dev. Cell, 2002, 2, 643). A transient Wnt signal provides the crucial initial stimulus for the start of a new hair growth cycle, by activating beta-catenin and TCF-regulated gene transcription in epithelial hair follicle precursors (Van Mater et al., Genes Dev., 2003, 17, 1219).
Because GSK-3 activity is associated with sperm motility, GSK-3 inhibition is useful as a male contraceptive. It was shown that a decline in sperm GSK-3 activity is associated with sperm motility development in bovine and monkey epididymis. (Vijayaraghavan et al., Biol. Reprod., 1996, 54, 709; Smith et al., J. Androl., 1999, 20, 47). Furthermore, tyrosine & serine/threonine phosphorylation of GSK-3 is high in motile compared to immotile sperm in bulls (Vijayaraghavan et al., Biol. Reprod., 2000, 62, 1647). This effect was also demonstrated with human sperm (Luconi et al., Human Reprod., 2001, 16, 1931).
Interleukin-1 receptor-associated kinase-4 (IRAK-4) is a 53 kDa member of the IRAK family of serine-threonine kinases. Within the family IRAK-4 and IRAK-1 appear to have finctional kinase domains while IRAK-2 and IRAK-m do not (Janssens S, Beyaert R., Mol. Cell. 2003 11, 293-302).
IRAK-4 is important for the innate and adaptive immune responses. It plays a major role in cellular responses to immune system modulators, finctioning in signal transduction from activated members of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily (Li, S. et al., Proc Natl. Acad. Sci. USA. 2002 99, 5567-5572; Suzuki, N., et al., J. Immunol. 2003 170, 4031-4035). IRAK-4 also has effects outside of the immune system such as influencing neurotrophin-driven neuronal survival (Mamidipudi, V. et al., J. Biol. Chem. 2002 277, 28010-28018). Upon binding proinflammatory cytokines IL-1 and IL-18 or pathogen-associated molecular pattern (PAMPs) ligands (eg. LPS, viral RNA, lipoproteins/peptidoglycans, etc.) their cognate receptors (IL-1R, IL-18R, and TLR receptor family, respectively) recruit a series of adaptors. IRAK-4 interacts with the resultant complex and propagates the activation signal through a series of additional proteins that ultimately stimulate IkappaB kinases (IKKs) and the mitogen-activated protein kinases (MAPKs), JNK and p38. These kinases stimulate NFkappaB- and AP-1-dependent transcription, the products of which are important for controlling processes such as cell survival and proinflammatory cytokine production (Yamamoto, Y. and Gaynor, R. B. et al., J. Clin. Invest. 2001 107, 135-142; Dunne, A. and O'Neill, L A J., Sci. STKE Feb. 25; 2003 (171):re3). Mice lacking IRAK-4 do not respond to IL-1 and ligands that stimulate various TLR's and are resistant to certain immunological challenges (Suzuki, N et al., Nature 2002 416, 750-756).
Activators of the IL-1R/TLR family contribute to a variety of diseases including inflammation and cancer (O'Neill L A., Sci STKE. Aug. 8; 2000 (44):RE1; Apte, R N. and Voronov, E., Semin, Cancer Biol. 2002 12, 277-290; Apte, R N. et al., Adv. Exp. Med. Biol. 2000 479, 277-88). IL-1 and IL-18 are important mediators of inflammatory diseases (Dinarello C A, Clin. Exp. Rheumatol. 2002: 20 (5 Suppl 27): S1-13) including rheumatoid arthritis (Dayer, J M, Rheumatology (Oxford), 2003 42, Suppl 2:ii3-10; Dai, S M., et al., Arthritis Rheum. 2004 50, 432-443) and inflammatory bowel disease (Lochner, M. and Forster, I., Pathobiology, 2002-2003 70, 164-169). TLR4 ligands such as LPS (O'Neill, L A J, Curr. Opin. Pharmacol. 2003 3, 396-403), hsp60 (Ohashi, K. et al., J. Immunol. 2000 164, 558-561), and fibronectin fragments (Okamura, Y. et al., J. Biol. Chem. 2001 276, 10229-10233) promote processes associated with the inflammatory response. Other diseases affected by TLR family may include autoimmunity (Eriksson, U., et al., Nat. Med. 2003 9, 1484-1490), viral infections (Vaidya, S. A. and Cheng, G., Curr. Opin. Immunol. 2003 15, 402-407; Tabeta K., et al., Proc. Natl. Acad. Sci. USA, 2004 101, 3516-3521; Diebold, S. S., et al., Science, 2004 303, 1529-1531), and sepsis (Cristofaro, P. and Opal, S. M. Expert Opin. Ther. Targets, 2003 7, 603-612).
The Janus kinases (JAK) are a family of tyrosine kinases consisting of JAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokine signaling. The down-stream substrates of the JAK family of kinases include the signal transducer and activator of transcription (STAT) proteins. JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis as well as in solid and hematologic malignancies such as leukemias and lymphomas. The pharmaceutical intervention in the JAK/STAT pathway has been reviewed [Frank, Mol. Med. 1999, 5, 432-456 and Seidel et al., Oncogene 2000, 19, 2645-2656].
JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 is predominantly expressed in hematopoietic cells. JAK3 binds exclusively to the common cytokine receptor gamma chain (γc) and is activated by IL-2, IL-4, IL-7, IL-9, and IL-15. The proliferation and survival of murine mast cells induced by IL-4 and IL-9 have, in fact, been shown to be dependent on JAK3- and γc-signaling [Suzuki et al., Blood 2000, 96, 2172-2180].
Cross-linking of the high-affinity immunoglobulin (Ig) E receptors of sensitized mast cells leads to a release of proinflammatory mediators, including a number of vasoactive cytokines resulting in acute allergic, or immediate (type I) hypersensitivity reactions [Gordon et al., Nature 1990, 346, 274-276 and Galli, N. Engl. J. Med. 1993, 328, 257-265]. A crucial role for JAK3 in IgE receptor-mediated mast cell responses in vitro and in vivo has been established [Malaviya et al., Biochem. Biophys. Res. Commun. 1999, 257, 807-813]. In addition, the prevention of type I hypersensitivity reactions, including anaphylaxis, mediated by mast cell-activation through inhibition of JAK3 has also been reported [Malaviya et al., J. Biol. Chem. 1999, 274, 27028-27038]. Targeting mast cells with JAK3 inhibitors modulated mast cell degranulation in vitro and prevented IgE receptor/antigen-mediated anaphylactic reactions in vivo.
A recent study described the successful targeting of JAK3 for immunosuppression and allograft acceptance. The study demonstrated a dose-dependent survival of Buffalo heart allograft in Wistar Furth recipients upon administration of inhibitors of JAK3 indicating the possibility of regulating unwanted immune responses in graft versus host disease [Kirken, Transpl. Proc. 2001, 33, 3268-3270].
IL-4-mediated STAT-phosphorylation has been implicated as the mechanism involved in early and late stages of rheumatoid arthritis (RA). Up-regulation of proinflammatory cytokines in RA synovium and synovial fluid is a characteristic of the disease. It has been demostrated that IL-4-mediated activation of IL-4/STAT pathway is mediated through the Janus Kinases (JAK 1 & 3) and that IL-4-associated JAK kinases are expressed in the RA synovium [Muller-Ladner et al., J. Immunol. 2000, 164, 3894-3901].
Familial amyotrophic lateral sclerosis (FALS) is a fatal neurodegenerative disorder affecting about 10% of ALS patients. The survival rates of FALS mice were increased upon treatment with a JAK3 specific inhibitor. This suggested that JAK3 plays a role in FALS [Trieu et al., Biochem. Biophys. Res. Commun. 2000, 267, 22-25].
Signal transducer and activator of transcription (STAT) proteins are activated by, among others, the JAK family kinases. Results from a recent study suggested the possibility of intervention in the JAK/STAT signaling pathway by targeting JAK family kinases with specific inhibitors for the treatment of leukemia [Sudbeck et al., Clin. Cancer Res. 1999, 5, 1569-1582]. JAK3 specific compounds were shown to inhibit the clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LC1-19, NALM-6, MOLT-3 and HL-60.
In animal models, TEL/JAK2 fusion proteins have induced myeloproliferative disorders and in hematopoietic cell lines, and introduction of TEL/JAK2 resulted in activation of STAT1, STAT3, STAT5, and cytokine-independent growth [Schwaller et al., EMBO J. 1998, 17, 5321-5333].
Inhibition of JAK3 and TYK2 abrogated tyrosine phosphorylation of STAT3, and inhibited cell growth of mycosis fungoides, a form of cutaneous T-cell lymphoma. These results implicated JAK family kinases in the constitutively activated JAK/STAT pathway that is present in mycosis fungoides [Nielsen et al., Proc. Nat. Acad. Sci. U.S.A. 1997, 94, 6764-6769]. Similarly, STAT3, STAT5, JAK1 and JAK2 were demonstrated to be constitutively activated in mouse T-cell lymphoma characterized initially by LCK over-expression, thus further implicating the JAK/STAT pathway in abnormal cell growth [Yu et al., J. Immunol. 1997, 159, 5206-5210]. In addition, IL-6-mediated STAT3 activation was blocked by an inhibitor of JAK, leading to sensitization of myeloma cells to apoptosis [Catlett-Falcone et al., Immunity 1999, 10, 105-115].
The c-met proto-oncogene encodes the Met receptor tyrosine kinase. The Met receptor is a 190 kDa glycosylated dimeric complex composed of a 50 kDa alpha chain disulfide-linked to a 145 kDa beta chain. The alpha chain is found extracellularly while the beta chain contains transmembrane and cytosolic domains. Met is synthesized as a precursor and is proteolytically cleaved to yield mature alpha and beta subunits. It displays structural similarities to semaphorins and plexins, a ligand-receptor family that is involved in cell-cell interaction. The ligand for Met is hepatocyte growth factor (HGF), a member of the scatter factor familyand has some homology to plaminogen [Longati, P. et al., Curr. Drug Targets 2001, 2, 41-55); Trusolino, L. and Comoglio, P. Nature Rev. Cancer 2002, 2, 289-300].
Met appears to be functioning in tumorigenesis and tumor metastasis. Chromosomal rearrangements forming Tpr-met fusions in an osteoclast cell line resulted in constitutively active Met receptors and transformation (Cooper, C. S. et al., Nature 1984, 311, 29-33). Met mutants exhibiting enhanced kinase activity have been identified in both hereditary and sporadic forms of papillary renal carcinoma (Schmidt, L. et al., Nat. Genet. 1997, 16, 68-73; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1997, 94, 11445-11500). Expression of Met along with its ligand HGF is transforming, tumorigenic, and metastatic (Jeffers, M. et al., Oncogene 1996, 13, 853-856; Michieli, P. et al., Oncogene 1999, 18, 5221-5231). HGF/Met has been shown to inhibit anoikis, suspension-induced programmed cell death (apoptosis), in head and neck squamous cell carcinoma cells. Anoikis resistance or anchorage-independent survival is a hallmark of oncogenic transformation of epithelial cells (Zeng, Q. et al., J. Biol. Chem. 2002, 277, 25203-25208).
HGF/Met signaling is involved in cell adhesion and motility in normal cells and plays a major role in the invasive growth that is found in most tissues, including cartilage, bone, blood vessels, and neurons (reviewed in Comoglio, P. M. and Trusolino, L. J. Clin. Invest. 2002, 109, 857-862). Dysfunctional activation or increased numbers of Met is likely to contribute to the aberrant cell-cell interactions that lead to migration, proliferation, and survival of cells that is characteristic of tumor metastasis. Activation of Met induces and sustains a variety of tumors [Wang, R. et al., J. Cell. Biol. 2001, 153, 1023-1034; Liang, T. J. et al., J. Clin. Invest. 1996, 97, 2872-2877; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1998, 95, 14417-14422] while loss of Met inhibits growth and invasiveness of tumor cells [Jiang, W. G. et al., Clin. Cancer Res. 2001, 7, 2555-2562; Abounader, R. et al., FASEB J. 2002 16, 108-110]. Increased expression of Met/HGF is seen in many metastatic tumors including colon (Fazekas, K. et al., Clin. Exp. Metastasis 2000, 18, 639-649), breast (Elliott, B. E. et al., 2002, Can. J. Physiol. Pharmacol. 80, 91-102), prostate (Knudsen, B. S. et al., Urology 2002, 60, 1113-1117), lung (Siegfried, J. M. et al., Ann. Thorac. Surg. 1998, 66, 1915-1918), and gastric (Amemiya, H. et al., Oncology 2002, 63, 286-296).
HGF-Met signaling has also been associated with increased risk of atherosclerosis (Yamamoto, Y. et al., J. Hypertens. 2001, 19, 1975-1979; Morishita, R. et al., Endocr. J. 2002, 49, 273-284) and increased fibrosis of the lung (Crestani, B. et al., Lab. Invest. 2002, 82, 1015-1022.
Syk is a tyrosine kinase that plays a critical role in FcεERI mediated mast cell degranulation and eosinophil activation. Accordingly, Syk kinase is implicated in various allergic disorders, in particular asthma. It has been shown that Syk binds to the phosphorylated gamma chain of the FcεRI receptor via N-terminal SH2 domains and is essential for downstream signaling [Taylor et al., Mol. Cell. Biol. 1995, 15, 4149].
Inhibition of eosinophil apoptosis has been proposed as a key mechanism for the development of blood and tissue eosinophilia in asthma. IL-5 and GM-CSF are upregulated in asthma and are proposed to cause blood and tissue eosinophilia by inhibition of eosinophil apoptosis. Inhibition of eosinophil apoptosis has been proposed as a key mechanism for the development of blood and tissue eosinophilia in asthma. It has been reported that Syk kinase is required for the prevention of eosinophil apoptosis by cytokines (using antisense) [Yousefi et al., J. Exp. Med. 1996, 183, 1407].
The role of Syk in FcγR dependent and independent response in bone marrow derived macrophages has been determined by using irradiated mouse chimeras reconstituted with fetal liver cells from Syk −/− embryos. Syk deficient macrophages were defective in phagocytosis induced by FcγR but showed normal phagocytosis in response to complement [Kiefer et al., Mol. Cell. Biol. 1998, 18, 4209]. It has also been reported that aerosolized Syk antisense suppresses Syk expression and mediator release from macrophages [Stenton et al., J. Immunology 2000, 164, 3790].
ZAP-70 is essential for T-cell receptor signalling. Expression of this tyrosine kinase is restricted to T-cells and natural killer cells. The importance of ZAP-70 in T-cell function has been demonstrated in human patients, human T-cell lines and mice. Human patients suffering from a rare form of severe combined deficiency syndrome (SCID) possess homozygous mutations in ZAP-70 (reviewed in Elder J. of Pedriatric Hematology/Oncology 1997, 19(6), 546-550). These patients have profound immunodeficiency, lack CD8+ T-cells and have CD4+ T-cells that are unresponsive to T-cell receptor (TCR)-mediated stimulation. Following TCR activation these CD4+ cells show severe defects in Ca2+ mobilization, tyrosine phosphorylation of down-stream substrates, proliferation and IL-2 production 70 (reviewed in Elder Pedriatric Research 39, 743-748). Human Jurkat cells lacking ZAP-70 also provide important insights into the critical role of ZAP-70 in T-cell receptor signalling. A Jurkat clone (p116) with no detectable ZAP-70 protein was shown to have defects in T-cell receptor signalling which could be corrected by re-introduction of wild type ZAP-70 (Williams et al., Molecular and Cellular Biology 1998, 18 (3), 1388-1399). Studies of mice lacking ZAP-70 also demonstrate a requirement of ZAP-70 in T-cell receptor signalling. ZAP-70-deficient mice have profound defects in T-cell development and T-cell receptor signalling in thymocytes is impaired (Negishi et al., Nature 1995 376, 435-438).
The importance of the kinase domain in ZAP-70 function is demonstrated by studies of human patients and mice expressing identical mutations in the DLAARN motif within the kinase domain of ZAP-70. Inactivation of kinase activity by this mutation results in defective T-cell receptor signalling (Elder et al., J. Immunology 2001, 656-661). Catalytically inactive ZAP-70 (Lys369Arg) was also defective in restoring T-cell receptor signalling in a ZAP-70 deficient Jurkat cell clone (p116) (Williams et al., Molecular and Cellular Biology 1998, 18 (3), 1388-1399).
Transforming growth factor-beta (TGF-beta) activated kinase 1 (TAK-1) is a 67 kDa ubiquitin-dependent serine-threonine kinase that functions as a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK or MEKK) (Wang, C., et al., Nature 2001, 412, 346-351).
Originally described as stimulated by TGF-beta superfamily members (Yamaguchi K. et al., Science 1995, 270, 2008-2011) TAK-1 is known to also function in signaling from numerous cell modulators including proinflammatory cytokines. TAK-1 is critical for signaling from IL-1beta/TLR ligands (Holtmann H, et al., J. Biol. Chem. 2001, 276, 3508-3516; Jiang Z, et al., J. Biol. Chem. 2003, 278, 16713-16719) and TNF-alpha (Takaesu G. et al., J. Mol. Biol. 2003, 326, 105-115). In addtion TAK-1 plays a role in IL-18 (Wald, D., et al., Eur. J. Immunol. 2001, 31, 3747-3754), RANKL (Mizukami J., et al., Mol. Cell. Biol. 2002, 22, 992-1000) and ceramide (Shirakabe K., et al., J. Biol. Chem. 1997, 272, 8141-8144) signaling.
Through interaction with corresponding cell surface receptors these ligands stimulate TAK-1 to relay signals to a variety of pathways such as IKK/NFkappaB, JNK, and p38, that are important regulators of cellular processes including apoptosis (Edlund S., et al., Mol Biol Cell. 2003, 14, 529-544), differentiation (Suzawa, M. et al., Nat Cell Biol 2003, 5, 224-230), and cell cycle progression (Bradham C A, et al., Am J Physiol Gastrointest Liver Physiol. 2001 281, G1279-89).
Modification of signaling pathways can alter cellular processes and contribute to disease. Due to its central role in signaling from numerous cell surface receptors TAK-1 may be an important therapeutic target for a variety of diseases. The cytokines IL-1beta and TNFalpha are important mediators of inflammation in rheumatoid arthritis and other inflammatory diseases (Maini R N. and Taylor P C. Ann. Rev. Med. 2000, 51, 207-229). TAK-1 may be important in regulating disease-relevant cellular responses in these cases (Hammaker D R, et al. J. Immunol. 2004, 172, 1612-1618). TAK-1 affects cellular fibrotic responses (Ono K., et al., Biochem. Biophys. Res. Commun. 2003, 307, 332-337). It may also plays a role in heart failure (Zhang, D., Nat. Med. 2000, 6, 556-563), osteoporosis (Mizukami J, et al., Mol. Cell. Biol. 2002, 22, 992-1000) and survival of hepatocellular carcinoma cells (Arsura M, et al. Oncogene 2003, 22, 412-425). TAK-1 signaling may affect neurite outgrowth (Yanagisawa M., et al. Genes Cells. 2001, 6, 1091-1099) and is involved in control of adipogenesis (Suzawa M., et al. Nat. Cell. Biol. 2003, 5, 224-230) and cardiomyocyte differentiation (Monzen K., et al. J. Cell. Biol. 2001, 153(4), 687-698.
Accordingly, there is a great need to develop compounds useful as inhibitors of protein kinases. In particular, it would be desirable to develop compounds that are useful as inhibitors of CDK-2, cMET, FLT-3, JAK-3, GSK-3, IRAK-4, SYK, p70S6K, TAK-1, and ZAP-70 particularly given the inadequate treatments currently available for the majority of the disorders implicated in their activation.